β-Adrenergic receptor-PI3K signaling crosstalk in mouse heart: elucidation of immediate downstream signaling cascades

Abstract

Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β(1)/β(2)AR double knockout mice. We further showed the specificity of β(2)AR in this crosstalk as treatment with formoterol, a β(2)AR-selective agonist, but not dobutamine, a predominantly β(1)AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR (the mammalian target of rapamycin), P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β(2)AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β(2)AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions.

Figure 1. Acute βAR stimulation in vivo increases cardiac PI3K activity and phophorylation of Akt and ERK1/2.

Adult male C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. (A) Left ventricular tissue lysates were IP with an anti-pY antibody and subjected to in vitro lipid kinase assay. PIP, the phosphorylated end-product. The bar graph shows the densitometric scanning results of the measurement of PI3K activities in the control (n = 6) and ISO-treated (n = 6) mice. (B, C) Representative Western blot analyses were performed on LV tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. The bar graphs show the densitometric scanning results from two individual experiments (n = 6). In another series of experiments, mice were pretreated with vehicle (5% DMSO) or LY294002 (LY, 1.4 mg/kg, i.p.) for 30 min before the ISO treatment. In vitro lipid kinase assay (D) and Western blot analyses (E) were performed as described above. The bar graph shows the densitometric scanning results in the control (n = 6), ISO (n = 6), and LY/ISO (n = 6) groups. In all Western blotting experiments, data were normalized with individual total protein levels. *, p<0.05 versus vehicle control.

Figure 2. The time course of the effect of βAR stimulation in vivo on phophorylation of Akt, ERK1/2, P70S6K, S6, GSK-3α/β and FOXO3a.

Adult male C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for the indicated time. Shown are representative Western blots performed on LV tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-ERK1/2 (Thr202/Tyr204), phospho-P70S6K (Thr389), phospho-P70S6K (Thr421/Ser424), phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), phospho-GSK-3α (Ser21), phospho-GSK-3β (Ser9), and phospho-FOXO3a (Ser318/321). Blots of individual total protein and actin were also included.

Figure 3. Acute βAR stimulation doesn't affect the activity of PI3K/Akt or ERK1/2 in β1/β2DKO mouse heart.

Adult male β₁/β₂ double knockout mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. (A) Left ventricular lysates were subjected to in vitro lipid kinase assay as described above. The bar graph shows the densitometric scanning results of the measurement of cardiac PI3K activities in the control (n = 3) and ISO-treated (n = 3) mice. (B, C) Representative Western blot analyses were performed on LV tissue lysates as described above. The bar graphs show the densitometric scanning results from three seperate experiments. Data are normalized with individual total protein levels.

Figure 4. Acute βAR stimulation induced increase in phosphorylation of Akt is cardiac-specific.

Adult male C57BL/6 mice were treated with vehicle control (saline) or isoproterenol (ISO, 1.25 mg/kg, i.N) for 30 min. (A) Representative Western blot analyses were performed on lung or kidney tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. (B, C) The bar graphs show the densitometric scanning results from two seperate experiments (n = 6). Data are normalized with individual total protein levels and represent means ± S.E. of percent change in protein phosphorylation relative to that of vehicle control. *, p<0.05 versus vehicle control.

Figure 5. Acute βAR stimulation induced-transactivation of PI3K signaling pathway is PKA-independent.

Adult male C57BL/6 mice were pretreated with vehicle (5% DMSO) or H-89 (20 mg/kg, i.p.) for 30 min before treatment with control (saline) or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. (A) In vitro lipid kinase assay was performed as described. PIP, the phosphorylated end-product. The bar graph shows the densitometric scanning results of the measurement of PI3K activities in the control, ISO-treated and H-89 + ISO-treated mice (n = 4). (B, C) Representative Western blot analyses were performed on LV tissue lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-FOXO1 (Thr24), total Akt and total FOXO. The bar graphs show the densitometric scanning results. Data are normalized with individual total protein levels and represent means ± S.E. of percent change in protein phosphorylation relative to that of the control. *, p<0.05 versus the control.

Figure 6. Acute βAR stimulation induced increase in phosphorylation of Akt and ERK1/2 is β2AR-specific.

C57BL/6 mice were treated with vehicle control, dobutamine (Dob., 1.7 mg/kg, i.p.), or formoterol (For., 2.1 mg/kg, i.p.), for 30 min. (A) In vitro lipid kinase assay was performed as described except with higher amount of LV lysates (1 mg). PIP, the phosphorylated end-product. The bar graph shows the densitometric scanning results of the measurement of PI3K activities in the control, Dob.-treated and For.-treated mice (n = 4). (B) Representative Western blot analyses were performed on left ventricular lysates with antibodies against phospho-Akt (Thr308), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. The bar graphs show the densitometric scanning results from two seperate experiments (n = 5). Data are normalized with individual total protein levels and represent means ± S.E. of percent change in protein phosphorylation relative to that of vehicle control. *, p<0.05 versus vehicle control.

Figure 7. Acute β2AR stimulation induced increases in phosphorylation of P70S6, S6, GSK3α, GSK3β and FOXO3a.

C57BL/6 mice were treated with vehicle control, dobutamine (Dob., 1.7 mg/kg, i.p.), or formoterol (For., 2.1 mg/kg, i.p.), for 30 min. Shown are representative Western blots performed on left ventricular lysates with antibodies against phospho-P70S6K (Thr389), phospho-P70S6K (Thr421/Ser424), phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), phospho-GSK-3α (Ser21), phospho-GSK-3β (Ser9), phospho-FOXO3a (Ser318/321). Blots of individual total protein and activ were also included.

Figure 8. Acute βAR stimulation in vivo induces increases in the phophorylation of mTOR, P70S6K and S6.

C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. Representative Western blot analyses were performed on left ventricular lysates with antibodies against (A) phospho-mTOR (Ser2448) and total mTOR; (B) phospho-P70S6K (Thr389, Thr421/Ser424) and total P70S6K; (C) phospho-S6 (Ser235/236, Ser240/244) and total S6. The bar graphs show the densitometric scanning results from two seperate experiments (n = 6). Data are normalized with individual total protein levels. *, p<0.05 versus vehicle control.

Figure 9. Acute βAR stimulation in vivo induces increases in the phophorylation of GSK-3α and GSK-3β.

C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. Representative Western blot analyses were performed on left ventricular lysates with antibodies against phospho-GSK-3α (Ser21), total GSK-3α, phospho-GSK-3β (Ser9), and total GSK-3β. The bar graphs show the densitometric scanning results from two seperate experiments (n = 6). Data are normalized with individual total protein levels. *, p<0.05 versus vehicle control.

Figure 10. Acute βAR stimulation in vivo induces an increase in the phophorylation of FOXO1 and FOXO3a.

C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, i.p.) for 30 min. Representative Western blot analyses were performed on left ventricular lysates with antibodies against phospho-FOXO1(Thr24)/FOXO3a(Thr32), phospho-FOXO3a (Ser318/321, Ser253), total FOXO1 and total FOXO3a. The bar graphs show the densitometric scanning results from two seperate experiments (n = 6). Data are normalized with individual total protein levels. *, p<0.05 versus vehicle control.

Figure 11. Acute βAR stimulation time-dependently decreases the levels of atrogin-1 and MuRF1 mRNA in mouse heart.

Adult male C57BL/6 mice were treated with vehicle control or isoproterenol (ISO, 1.25 mg/kg, I.p.) for up to six hours (A). In a separate experiment, mice were treated with vehicle control or ISO for four hours. The mRNA levels of atrogin-1 (B, n = 3) and MuRF1 (C, n = 3) were determined by real time quantitative PCR using specific TaqMan probes and normalized to GAPDH. The plot lines or bar graphs represent the means ± S.E of percent change in mRNA level relative to that of vehicle control. *, p<0.05 versus vehicle control.

Figure 12. A hypothetical signaling cascades for the crosstalk between β2AR and PI3K/Akt pathway in vivo.

Activation of ERK1/2-related signaling and the mTOR/P70S6K/S6 axis favors the progression of cardiac hypertrophy. Akt-induced phosphyrylation of GSK-3α/β inhibits its activity, thereby nullifying its anti-hypertrophic effect. Akt-induced phosphorylation of FOXOs also inhibits ist activity, negating its effects on activation of atrogin-1 and MuRF1, which results in decreased protein breakdown (atrophy). These multiple signaling scenarios likely contribute to the pathogenesis of cardiac hypertrophy and/or the anti-atrophic effect following sustained βAR stimulation. “→” represents activation, “⊣” represents inhibition. Solid lines represent data supplied by our study, and dotted lines represent hyperthetical schematics.